Elevated Levels Of Oxygen Research Articles

The oxidative stress mechanism of Bacillus cereus spores induced by atmospheric pressure plasma jet

Emerging plasma technology has demonstrated its effectiveness in inactivating bacteria and spores, showing wide applicability in environment cleaning and in the food supply chain. Studies have revealed that the main reason of plasma-mediated bacterial inactivation were contributed to the oxidative damage on inner membranes (IMs) of cell, while few research paid attention to the oxidation stress process of lipid in spores’ IMs. This study investigated the impact of atmospheric pressure plasma jet (APPJ) treatment on spore inactivation, focusing on oxidative stress mechanisms and changes in membrane lipid composition. Results demonstrated that APPJ treatment induced significant oxidative stress within spores, characterized by elevated levels of reactive oxygen and nitrogen species (RONS). This oxidative stress led to the peroxidation of glycerophospholipids, particularly phosphatidylglycerol, phosphatidylcholine, and cardiolipin, major components of spore membranes. Subsequently, the rearrangement of membrane lipid and the formation of lipid hydroperoxide, caused by the released cations and RONS, accelerated membrane instability. These changes correlated with increased membrane permeability and structural damage observed via flow cytometry, indicating the critical role of membrane lipid peroxidation in spore inactivation. Furthermore, the study highlighted the potential of APPJ as a robust technology for enhancing food safety by targeting spore-forming pathogens throughout the food supply chain.

Scanning Electron Microscopy and Energy-Dispersive X-ray Spectroscopy of Staphylococcus aureus Biofilms.

Understanding the dynamics of biofilm formation and its elemental composition is crucial for developing effective strategies against biofilm-associated infections. In this study, we employed scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) to investigate the morphological changes and elemental compositions of Staphylococcus aureus biofilms. SEM images revealed distinct stages of biofilm development, from initial aggregation to the formation of mature and aged biofilms. EDS analysis consistently showed elevated levels of sodium (Na), oxygen (O), and phosphorus (P) in the biofilm matrix, indicating its high negative charge and the presence of anionic biopolymers. The incorporation of extracellular DNA (eDNA) into the biofilm matrix, leading to significant retention of sodium ions, underscored the importance of electrostatic interactions in biofilm formation and stability. Our findings highlight the potential of EDS analysis in quantifying elemental compositions and elucidating the role of anionic biopolymers in biofilm development.

Comparison of 2D and 3D oxygen-enhanced MRI of the placenta.

Oxygen-Enhanced Magnetic Resonance Imaging (OE-MRI) of the human placenta is potentially a sensitive marker of in vivo oxygenation. This methodological study shows that full coverage of the placenta is possible using 3D mapping of the change in longitudinal relaxation rate (ΔR1), in a group of healthy pregnant subjects breathing elevated levels of oxygen. Twelve pregnant subjects underwent a comparison of 2D and 3D OE-MRI. ΔR1 was mapped for a single 2D slice (ss-2D), a single matched-slice from the 3D volume (ss-3D) and the full 3D volume (vol-3D). The group-average median ΔR1 values for ss-3D (0.023 s-1) and vol-3D (0.022 s-1) do not differ significantly from ss-2D (0.020 s-1), when compared using a two-tailed paired t-test (ss-3D (p = 0.58) and vol-3D (p = 0.70)). However, median baseline T1 (T1b) for ss-2D was higher (1603 ms) than T1b for ss-3D (1540 ms, p = 0.07) and significantly higher than vol-3D (1515 ms, p = 0.02), when compared using a two-tailed paired t-test. In contrast with previous studies, no correlation of median ΔR1 with gestation age at scan for the normal group (N = 10) was observed for ss-2D, likely due to the smaller gestational range. Full volume OE-MRI maps reveal sensitivity to changes in ΔR1, with some participants showing an enhanced gradient in the intermediate space between the fetal and maternal sides of the placenta in the 3D data. This study shows that it is feasible to acquire whole placental volume OE-MRI data in women with healthy pregnancy.

Cerium oxide-modified Pd nanosheets encapsulated by red blood cell membranes for high-efficiency RONS scavenging in depression treatment.

Depression, a common and high-morbidity mental illness, can cause severe physical and psychiatric disorder. Recently, elevated levels of reactive oxygen and nitrogen species (RONS) have been recognized as a potential pathogenesis of depression. Unfortunately, available therapies provide limited outcomes in treating RONS-related depression symptoms. In addition, the low blood-brain barrier (BBB) penetration efficiency of some drugs is another major barrier to depression treatment. This study developed cerium oxide-modified Pd (Pd-CeO2) nanosheets with outstanding antioxidant activities for depression therapy. Under physiological conditions, Pd-CeO2 exhibited significant O2˙- and H2O2 clearance through their superoxide dismutase (SOD) and catalase (CAT) activities. Meanwhile, Pd-CeO2 also displayed the ability to scavenge ˙OH and reactive nitrogen radicals (RNS). What's more, when incorporated with biocompatible red blood cell (RBC) membranes, Pd-CeO2@RBC could overcome the BBB and protect brain tissues from oxidative damage caused by RONS. As a result, Pd-CeO2@RBC therapy reduced the proliferation of microglia and astrocytes and alleviated neuroinflammation and depression-like behaviors. This research not only provides a novel strategy for the effective treatment of depression, but also paves the way for new therapeutic options of nanozymes in neurological disorders.

Effects on Pulmonary Functions in Patients Undergoing Hyperbaric Oxygen Therapy: A Prospective Observational Study

Abstract Introduction: Hyperbaric Oxygen Therapy (HBOT) consists of administering oxygen at pressures higher than atmospheric pressure. Though HBOT remains one of the safest therapies used today, it is not completely devoid of side effects. Pulmonary oxygen toxicity can result as a result of continuous exposure of elevated levels of oxygen for increased duration and at increased pressures. The main pathological manifestations occur as acute exudative phase followed by subacute proliferative phase. Effects of HBOT on pulmonary functions has not been studied in detail. This study was conducted to ascertain the effects of HBOT on pulmonary functions and determine if these changes are reversible in nature. Material and Method: A prospective observational study was conducted at a tertiary care hospital over a period of 02 years. A total of 72 patients who were undergoing HBOT for any indication normal chest radiograph and normal spirometry/DLCO were included in the study. Spirometry and DLCO was done initially at the time of inclusion and repeated after half of the total planned HBOT sessions and finally at the end of all sessions of HBOT. Spirometry and DLCO was also repeated 6 months post completion of HBOT. The HBOT protocol consisted of breathing 100% oxygen at 2.4 atmospheric absolute (ATA) for 90 minutes which was referred as one session. Total sessions varied from 20-30 based on indication and response of their primary disease to HBO. Results: The mean age score in study population was 47.12 ± 12.88. Among the study population, 12(24%) participants were male and 38(76%) participants were female. The mean BMI was 24.49 ± 5.45. The mean DLCO was 6.67 ± 1.45 ml/min/mm Hg pre intervention, it was 6.38 ± 1.43 ml/min/mm Hg at mid cycle and 6.21 ± 1.41 ml/min/mm Hg post intervention and at 6 months follow up it was 6.43 ± 1.46 ml/min/mm Hg. The difference in the DLCO at mid cycle, post intervention and 6 months follow up period with baseline value were statistically significant (P value <0.05). The mean FVC was 2.59 ± 0.59 L pre intervention, 2.41 ± 0.6 L at mid cycle, 2.22 ± 0.53 L immediately post intervention and 2.44 ± 0.59 L at 6 months follow up. The difference in the FVC score at mid cycle, post intervention and 6 months follow up with baseline value were statistically significant (P value <0.001). The mean FEV1 was 2.15 ± 0.52L pre- intervention, 2.14 ± 1.97L at mid cycle, 2.03 ± 0.53 L post intervention and 2.52 ± 2.02 L at 6 months follow up. The difference in the FEV1 score at post intervention with baseline value (pre-intervention) was statistically significant (P value <0.001). However, the difference in the FEV1 score at 6 months follow up period with baseline value (pre-intervention) was not statistically significant (P value >0.005). Conclusion: HBOT although a safe intervention, it is not totally devoid of complications. Although our study did reveal statistically significant changes in pulmonary function in most of the instances, it did not show any clinically significant changes in pulmonary function with use of HBOT protocol at 2.4 ATA. Studies with larger sample size will be required to ensure evidence based safety of HBOT use.

Hyperbaric Oxygen and Outcomes Following the Brain Injury: A Systematic Review.

Hyperbaric oxygen therapy (HBO2) aims to address ischemia resulting from brain injury by subjecting patients to an atmosphere that dramatically raises the concentration of inspired oxygen (100% O2 at greater than 1 ATA). This results in elevated levels of oxygen in the plasma, which in turn boosts the delivery of oxygen for diffusion to the brain tissue. To study the efficacy of hyperbaric oxygen (HBO)-based modalities in brain injury. Preferred reporting items for systematic reviews protocol was applied to perform literature search regarding this analytical review. In our study, fifteen studies are included in this review, involving 1067 people. The mean age group of patients enrolled was 57.0±11.6 and the mean NIHSS score was 10.5±8.7, of which 21 participants had moderate to severe neurological impairment. The total number of HBO treatments was 8 to 70 times (28.3±17.9), at the end of the 6-month follow-up period. mRS (modified Rankin scale) ≤3 was found in 25 cases, of which 12 patients with high-grade aSAH recovered. Poor prognosis was prevalent in patients who experienced delayed cerebral ischemia, this was true for 22.7% of patients in this study. In 3 studies conducted by Rockswold, ICP (mm Hg) was significantly lower in the HBO2 group after the treatment than pretreatment. (p<0.05). 4 studies showed an improvement in GCS score after HBO2 therapy.One trial (Imai 2006) reported that three patients in the HBO group died due to pneumonia (two) and heart failure (one) and one patient died in the control group due to heart failure. Overall, it is relatively safe to use HBO in the treatment of brain-related haemorrhage, strokes, and injury as there were no major complications reported. This systematic review demonstrates that HBO2 has significant clinical potential in treatment of brain related haemorrhages, stroke and injury.

Endothelial progenitor cells derived from embryonic stem cells prevent alveolar simplification in a murine model of bronchopulmonary dysplasia.

Introduction: Vascular remodeling and compromised alveolar development are hallmarks of chronic pulmonary diseases such as bronchopulmonary dysplasia (BPD). Despite advances in neonatal healthcare the number of BPD cases worldwide continues to increase. One approach to overcoming the premature arrest in lung development seen in BPD is to stimulate neonatal angiogenesis via delivery and engraftment of endothelial progenitor cells (EPCs). One such population is resident to the pulmonary microvasculature and expresses both FOXF1 and c-KIT. Previous studies have shown that c-KIT+FOXF1+ EPCs are highly sensitive to elevated levels of oxygen (hyperoxia) and are decreased in premature infants with BPD and hyperoxia-induced BPD mouse models. We hypothesize that restoring EPCs through transplantation of c-KIT+FOXF1+ EPCs derived in vitro from pluripotent embryonic stem cells (ESCs), will stimulate neonatal angiogenesis and alveolarization in mice with hyperoxia-induced lung injury. Methods: Utilizing a novel ESC line with a FOXF1:GFP reporter, we generated ESC-derived c-KIT+FOXF1+ EPCs in vitro. Using a second ESC line which contains FOXF1:GFP and tdTomato transgenes, we differentiated ESCs towards c-KIT+FOXF1+ EPCs and tracked them in vivo after injection into the neonatal circulation of hyperoxia-injured mice. After a recovery period in room air conditions, we analyzed c-KIT+FOXF1+ EPC engraftment and quantified the number of resident and circulating endothelial cells, the size of alveolar spaces, and the capillary density after EPC transplantations. Results and conclusion: Herein, we demonstrate that addition of BMP9 to the directed endothelial differentiation protocol results in very efficient generation of c-KIT+FOXF1+ EPCs from pluripotent ESCs. ESC-derived c-KIT+FOXF1+ EPCs effectively engraft into the pulmonary microvasculature of hyperoxia-injured mice, promote vascular remodeling in alveoli, increase the number of resident and circulating endothelial cells, and improve alveolarization. Altogether, these results provide a proof-of-principle that cell therapy with ESC-derived c-KIT+FOXF1+ EPCs can prevent alveolar simplification in a hyperoxia-induced BPD mouse model.

Fully-resolved simulations of volatile combustion and NO[formula omitted] formation from single coal particles in recycled flue gas environments

The interaction of coal particles and recycled/recirculated flue gas (RFG) with elevated temperatures and low levels of oxygen occurs in various pulverised coal combustion scenarios. In this work, the effect of oxygen level and temperature on single coal particle combustion characteristics and NOx formation in N2 diluent is studied by means of fully-resolved particle simulations. Comprehensive gas-phase kinetics are utilised to consider the critical pathways of NOx formation including tar-N. Results show that higher RFG temperatures decrease the time to reach the peaks of temperature and species profiles and increase the corresponding peak values. When decreasing O2, irrespective of the RFG temperature, the fuel release period is prolonged, the volatile combustion time increases and the combustion process becomes overall less intense. The reduction of O2 in RFG results in a significant decrease of NO production, while the reduction of the RFG temperature has a smaller effect. The analysis of the key reactions that contribute to NO production in the region around stoichiometry shows that fuel-NOx is the major contributor. Both NH3 and HCN in fuel-N play a major role, while tar-N only contributes in the case with the lowest temperature and O2 concentration. The classical NOx formation pathways are negligible and the initiation reaction of the Zeldovich mechanism is even reversed, i.e. NO⟶+NN2 is dominant and contributes to NO destruction. The destruction of NO mainly occurs in a rich region close to the particle surface where abundant tar species and their derivatives play a major role for NO destruction via the re-burn mechanism. The prompt mechanism is also active in this region and eventually contributes to NO reduction via production of HCN which is the feed to the re-burn mechanism.

Vibrio cholerae requires oxidative respiration through the bd-I and cbb3 oxidases for intestinal proliferation.

Vibrio cholerae respires both aerobically and anaerobically and, while oxygen may be available to it during infection, other terminal electron acceptors are proposed for population expansion during infection. Unlike gastrointestinal pathogens that stimulate significant inflammation leading to elevated levels of oxygen or alternative terminal electron acceptors, V. cholerae infections are not understood to induce a notable inflammatory response. To ascertain the respiration requirements of V. cholerae during infection, we used Multiplex Genome Editing by Natural Transformation (MuGENT) to create V. cholerae strains lacking aerobic or anaerobic respiration. V. cholerae strains lacking aerobic respiration were attenuated in infant mice 105-fold relative to wild type, while strains lacking anaerobic respiration had no colonization defect, contrary to earlier work suggesting a role for anaerobic respiration during infection. Using several approaches, including one we developed for this work termed Comparative Multiplex PCR Amplicon Sequencing (CoMPAS), we determined that the bd-I and cbb3 oxidases are essential for small intestinal colonization of V. cholerae in the infant mouse. The bd-I oxidase was also determined as the primary oxidase during growth outside the host, making V. cholerae the only example of a Gram-negative bacterial pathogen in which a bd-type oxidase is the primary oxidase for energy acquisition inside and outside of a host.

Pathogenesis of Multiple Organ Failure: The Impact of Systemic Damage to Plasma Membranes.

Multiple organ failure (MOF) is the major cause of morbidity and mortality in intensive care patients, but the mechanisms causing this severe syndrome are still poorly understood. Inflammatory response, tissue hypoxia, immune and cellular metabolic dysregulations, and endothelial and microvascular dysfunction are the main features of MOF, but the exact mechanisms leading to MOF are still unclear. Recent progress in the membrane research suggests that cellular plasma membranes play an important role in key functions of diverse organs. Exploration of mechanisms contributing to plasma membrane damage and repair suggest that these processes can be the missing link in the development of MOF. Elevated levels of extracellular phospholipases, reactive oxygen and nitrogen species, pore-forming proteins (PFPs), and dysregulation of osmotic homeostasis occurring upon systemic inflammatory response are the major extracellular inducers of plasma membrane damage, which may simultaneously operate in different organs causing their profound dysfunction. Hypoxia activates similar processes, but they predominantly occur within the cells targeting intracellular membrane compartments and ultimately causing cell death. To combat the plasma membrane damage cells have developed several repair mechanisms, such as exocytosis, shedding, and protein-driven membrane remodeling. Analysis of knowledge on these mechanisms reveals that systemic damage to plasma membranes may be associated with potentially reversible MOF, which can be quickly recovered, if pathological stimuli are eliminated. Alternatively, it can be transformed in a non-resolving phase, if repair mechanisms are not sufficient to deal with a large damage or if the damage is extended to intracellular compartments essential for vital cellular functions.

The Venom of Philippine Tarantula (Theraphosidae) Contains Peptides with Pro-Oxidative and Nitrosative-Dependent Cytotoxic Activities against Breast Cancer Cells (MCF-7) In Vitro.

Background:Breast cancer is a multifactorial disease that affects women worldwide. Its progression is likely to be executed by oxidative stress wherein elevated levels of reactive oxygen and nitrogen species drive several breast cancer pathologies. Spider venom contains various pharmacological peptides which exhibit selective activity to abnormal expression of ion channels on cancer cell surface which can confer potent anti-cancer activities against this disease. Methods:Venom was extracted from a Philippine tarantula by electrostimulation and fractionated by reverse phase-high performance liquid chromatography (RP-HPLC). Venom fractions were collected and used for in vitro analyses such as cellular toxicity, morphological assessment, and oxidative stress levels. Results:The fractionation of crude spider venom generated several peaks which were predominantly detected spectrophotometrically and colorimetrically as peptides. Treatment of MCF-7 cell line of selected spider venom peptides induced production of several endogenous radicals such as hydroxyl radicals (•OH), nitric oxide radicals (•NO), superoxide anion radicals (•O2−) and lipid peroxides via malondialdehyde (MDA) reaction, which is comparable with the scavenging effects afforded by 400 µg/mL vitamin E and L-cysteine (p<0.05). Concomitantly, the free radicals produced decrease the mitochondrial membrane potential and metabolic activity as detected by rhodamine 123 and tetrazolium dye respectively (p>0.05). This is manifested by cytotoxicity in MCF-7 cells as seen by increase in membrane blebbing, cellular detachment, caspase activity and nuclear fragmentation. Conclusion:These data suggest that the Philippine tarantula venom contains peptide constituents exhibiting pro-oxidative and nitrosative-dependent cytotoxic activities against MCF-7 cells and can indicate mechanistic insights to further explore its potential application as prooxidants in cancer therapy.

Abstract 4803: Limiting heme availability as an effective strategy to target lung cancer growth and progression in vivo

Abstract Lung cancer is the leading cause of cancer-related death in the US. About 85% of cases are non-small cell lung cancer (NSCLC). Several chemotherapeutic and targeted therapeutic agents are approved for treating lung cancer, but the five-year survival rate remains low. Thus, alternative approaches for are required for treatment of lung cancer. Several studies demonstrate that enhanced mitochondrial respiration or oxidative phosphorylation (OXPHOS) is a key feature of NSCLC. Heme is a central metabolic molecule that serves as a prosthetic group in several proteins involved in oxygen transport, utilization, and storage. Previous studies from our lab show elevated levels of heme synthesis, uptake, and oxygen utilizing hemoproteins in NSCLC cells compared to normal cells. The results also demonstrate that inhibiting heme synthesis selectively inhibits proliferation of NSCLC cells. We hypothesize that elevated levels of heme are used as fuel for enhanced OXPHOS by NSCLC cells. Therefore, targeting heme flux and function can be an effective way of targeting NSCLC growth and progression. If elevated heme metabolism is crucial for the tumorigenic functions of NSCLC cells, limiting heme availability may be effective for suppressing lung tumor growth and progression. Therefore, we tried to lower heme availability by utilizing bacterial hemophores. We designed several heme-targeting peptides (HTAs) and tested their ability to inhibit heme uptake in NSCLC cell lines, subcutaneous xenografts, and lung orthotopic xenografts. NSCLC cells expressing luciferase were used to implant subcutaneous xenografts or lung orthotopic xenografts in NOD/SCID mice. The mice were treated with HTA to check if limiting heme availability would delay growth in NSCLC tumor xenografts. Also, mice with lung orthotopic xenografts treated with HTA were treated with a well-known chemotherapeutic agent, docetaxel, to determine if HTA pre-treatment improves survival in these mice. In vivo bioluminiscence imaging (BLI) was used to analyze growth and progression of orthotopic lung xenografts. Our BLI data show that HTA treatment causes a significant decrease in radiance (total photons/second) in subcutaneous xenografts and lung orthotopic xenografts. This is supported by Hematoxylin and Eosin (H &amp; E) staining in paraffin-embedded tissue sections from lung orthotopic xenografts and tumor volumes of subcutaneous xenograft tumors resected from mice. Furthermore, in mice with lung orthotopic xenografts, HTA pre-treatment leads to a marked improvement in survival of mice further treated with docetaxel as compared to docetaxel treatment alone. Our results indicate that limiting heme availability can be an effective strategy to limit growth and progression of NSCLC and can be used to increase their vulnerability to chemotherapeutic agents like docetaxel. Citation Format: Poorva Ghosh, Sarada Preeta Kalainayakan, Sanchareeka Dey, Adnin Ashrafi, Li Zhang. Limiting heme availability as an effective strategy to target lung cancer growth and progression in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4803.

Mechanics and complications of reverse shoulder arthroplasty: morse taper failure analysis and prospective rectification

Since Sir John Charnley began his monumental hip arthroplasty work in 1958, clinical researchers have been incrementally improving longevity and functionality of total joint systems, although implant failure occurs on occasion. The purpose of this study is to report the fracture of the humeral tray Morse taper of a reverse total shoulder system (RTSS), which to date has not been reported with metallurgic analysis for any RTSS. There was no reported antecedent fall, motor vehicle collision, or other traumatic event prior to implant fracture in this case. Analysis was performed on the retrieved failed implant by Scanning Electron Microscopy (SEM) and Electron Dispersion Spectroscopy (EDS) in an attempt to determine the failure method, as well as to offer improvements for future implants. At the time of revision surgery all explants were retained from the left shoulder of a 61-year old male who underwent a non-complicated RTSS 4 years prior. The explants, particularly the cracked humeral tray, were processed as required for SEM and EDS. Analysis was performed on the failure sites in order to determine the chemical composition of the different parts of the implant, discover the chemical composition of the filler metal used during the electron beam welding process, and to detect any foreign elements that could suggest corrosion or other evidence of failure etiology. Gross visual inspection of all explants revealed that implant failure was a result of dissociation of the taper from the humeral tray at the weld, leaving the Morse taper embedded in the humeral stem while the tray floated freely in the patient’s shoulder. SEM further confirmed the jagged edges noted grossly at the weld fracture site, both suggesting failure due to torsional forces. EDS detected elevated levels of carbon and oxygen at the fracture site on the taper only and not on the humeral tray. In order to determine the origin of the high levels of C and O, it was considered that in titanium alloys, C and O are used as stabilizers that help raise the temperature at which titanium can be cast. Since the presence of stabilizers reduces ductility and fatigue strength, all interstitial elements are removed after casting. Considering this, the presence of C and O suggests that not all of the interstitials were removed during the manufacturing process, resulting in decreased fatigue strength. Further destructive analytical testing would verify weld quality and failure mode. RTSSs are quite successful in select patients not amenable to traditional shoulder arthroplasty options. This case report highlights how an implant may function well for several years and then suddenly fail without warning. SEM and EDS analysis suggest that residual C and O in the taper lowered the metal implant’s integrity, leading to torsional cracking at the weld junction of the humeral tray and the taper. The elevated levels of C and O measured at fracture sites on both the tray and the taper suggest poor quality filler metal or failure to remove all interstitial elements after casting. In both cases, the results would be decreased fatigue strength and overall toughness, leading to mechanical failure. A manufacturer’s recall of all implants soon followed the reporting of this implant failure; subsequently, the metal materials were changed from Ti6Al4V to both titanium alloy and cobalt-chrome alloy (Co-Cr-Mo). Time will tell if the alterations were sufficient.

Tamoxifen dosing for Cre-mediated recombination in experimental bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is the most common complication of preterm birth characterized by blunted post-natal lung development. BPD can be modelled in mice by exposure of newborn mouse pups to elevated oxygen levels. Little is known about the mechanisms of perturbed lung development associated with BPD. The advent of transgenic mice, where genetic rearrangements can be induced in particular cell-types at particular time-points during organogenesis, have great potential to explore the pathogenic mechanisms at play during arrested lung development. Many inducible, conditional transgenic technologies available rely on the application of the estrogen-receptor modulator, tamoxifen. While tamoxifen is well-tolerated and has been widely employed in adult mice, or in healthy developing mice; tamoxifen is not well-tolerated in combination with hyperoxia, in the most widely-used mouse model of BPD. To address this, we set out to establish a safe and effective tamoxifen dosing regimen that can be used in newborn mouse pups subjected to injurious stimuli, such as exposure to elevated levels of environmental oxygen. Our data reveal that a single intraperitoneal dose of tamoxifen of 0.2mg applied to newborn mouse pups in 10μl Miglyol vehicle was adequate to successfully drive Cre recombinase-mediated genome rearrangements by the fifth day of life, in a murine model of BPD. The number of recombined cells was comparable to that observed in regular tamoxifen administration protocols. These findings will be useful to investigators where tamoxifen dosing is problematic in the background of injurious stimuli and mouse models of human and veterinary disease.

Metabolic adaptations of Azospirillum brasilense to oxygen stress by cell-to-cell clumping and flocculation.

The ability of bacteria to monitor their metabolism and adjust their behavior accordingly is critical to maintain competitiveness in the environment. The motile microaerophilic bacterium Azospirillum brasilense navigates oxygen gradients by aerotaxis in order to locate low oxygen concentrations that can support metabolism. When cells are exposed to elevated levels of oxygen in their surroundings, motile A. brasilense cells implement an alternative response to aerotaxis and form transient clumps by cell-to-cell interactions. Clumping was suggested to represent a behavior protecting motile cells from transiently elevated levels of aeration. Using the proteomics of wild-type and mutant strains affected in the extent of their clumping abilities, we show that cell-to-cell clumping represents a metabolic scavenging strategy that likely prepares the cells for further metabolic stresses. Analysis of mutants affected in carbon or nitrogen metabolism confirmed this assumption. The metabolic changes experienced as clumping progresses prime cells for flocculation, a morphological and metabolic shift of cells triggered under elevated-aeration conditions and nitrogen limitation. The analysis of various mutants during clumping and flocculation characterized an ordered set of changes in cell envelope properties accompanying the metabolic changes. These data also identify clumping and early flocculation to be behaviors compatible with the expression of nitrogen fixation genes, despite the elevated-aeration conditions. Cell-to-cell clumping may thus license diazotrophy to microaerophilic A. brasilense cells under elevated oxygen conditions and prime them for long-term survival via flocculation if metabolic stress persists.

Potential molecular mechanisms underlying muscle fatigue mediated by reactive oxygen and nitrogen species.

Intense contractile activity causes a dramatic decline in the force and velocity generating capacity of skeletal muscle within a few minutes, a phenomenon that characterizes fatigue. Much of the research effort has focused on how elevated levels of the metabolites of ATP hydrolysis might inhibit the function of the contractile proteins. However, there is now growing evidence that elevated levels of reactive oxygen and nitrogen species (ROS/RNS), which also accumulate in the myoplasm during fatigue, also play a causative role in this type of fatigue. The most compelling evidence comes from observations demonstrating that pre-treatment of intact muscle with a ROS scavenger can significantly attenuate the development of fatigue. A clear advantage of this line of inquiry is that the molecular targets and protein modifications of some of the ROS scavengers are well-characterized enabling researchers to begin to identify potential regions and even specific amino acid residues modified during fatigue. Combining this knowledge with assessments of contractile properties from the whole muscle level down to the dynamic motions within specific contractile proteins enable the linking of the structural modifications to the functional impacts, using advanced chemical and biophysical techniques. Based on this approach at least two areas are beginning emerge as potentially important sites, the regulatory protein troponin and the actin binding region of myosin. This review highlights some of these recent efforts which have the potential to offer uniquely precise information on the underlying molecular basis of fatigue. This work may also have implications beyond muscle fatigue as ROS/RNS mediated protein modifications are also thought to play a role in the loss of muscle function with aging and in some acute pathologies like cardiac arrest and ischemia.

The many roads to mitochondrial dysfunction in neuroimmune and neuropsychiatric disorders.

BackgroundMitochondrial dysfunction and defects in oxidative metabolism are a characteristic feature of many chronic illnesses not currently classified as mitochondrial diseases. Examples of such illnesses include bipolar disorder, multiple sclerosis, Parkinson’s disease, schizophrenia, depression, autism, and chronic fatigue syndrome.DiscussionWhile the majority of patients with multiple sclerosis appear to have widespread mitochondrial dysfunction and impaired ATP production, the findings in patients diagnosed with Parkinson’s disease, autism, depression, bipolar disorder schizophrenia and chronic fatigue syndrome are less consistent, likely reflecting the fact that these diagnoses do not represent a disease with a unitary pathogenesis and pathophysiology. However, investigations have revealed the presence of chronic oxidative stress to be an almost invariant finding in study cohorts of patients afforded each diagnosis. This state is characterized by elevated reactive oxygen and nitrogen species and/or reduced levels of glutathione, and goes hand in hand with chronic systemic inflammation with elevated levels of pro-inflammatory cytokines.SummaryThis paper details mechanisms by which elevated levels of reactive oxygen and nitrogen species together with elevated pro-inflammatory cytokines could conspire to pave a major road to the development of mitochondrial dysfunction and impaired oxidative metabolism seen in many patients diagnosed with these disorders.

A novel model of chronic wounds: importance of redox imbalance and biofilm-forming bacteria for establishment of chronicity.

Chronic wounds have a large impact on health, affecting ∼6.5 M people and costing ∼$25B/year in the US alone [1]. We previously discovered that a genetically modified mouse model displays impaired healing similar to problematic wounds in humans and that sometimes the wounds become chronic. Here we show how and why these impaired wounds become chronic, describe a way whereby we can drive impaired wounds to chronicity at will and propose that the same processes are involved in chronic wound development in humans. We hypothesize that exacerbated levels of oxidative stress are critical for initiation of chronicity. We show that, very early after injury, wounds with impaired healing contain elevated levels of reactive oxygen and nitrogen species and, much like in humans, these levels increase with age. Moreover, the activity of anti-oxidant enzymes is not elevated, leading to buildup of oxidative stress in the wound environment. To induce chronicity, we exacerbated the redox imbalance by further inhibiting the antioxidant enzymes and by infecting the wounds with biofilm-forming bacteria isolated from the chronic wounds that developed naturally in these mice. These wounds do not re-epithelialize, the granulation tissue lacks vascularization and interstitial collagen fibers, they contain an antibiotic-resistant mixed bioflora with biofilm-forming capacity, and they stay open for several weeks. These findings are highly significant because they show for the first time that chronic wounds can be generated in an animal model effectively and consistently. The availability of such a model will significantly propel the field forward because it can be used to develop strategies to regain redox balance that may result in inhibition of biofilm formation and result in restoration of healthy wound tissue. Furthermore, the model can lead to the understanding of other fundamental mechanisms of chronic wound development that can potentially lead to novel therapies.

Intracellular chromium localization and cell physiological response in the unicellular alga Micrasterias

Various contaminants like metals and heavy metals are constantly released into the environment by anthropogenic activities. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. Chromium can cause harm to cell metabolism and development, when it is taken up by plants instead of necessary micronutrients such as for example iron. The uptake of Cr VI into plant cells has been reported to be an active process via carriers of essential anions, while the cation Cr III seems to be taken up inactively. Micrasterias denticulata, an unicellular green alga of the family Desmidiaceae is a well-studied cell biological model organism. Cr III and VI had inhibiting effects on its cell development, while cell division rates were only impaired by Cr VI. Transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization, condensed cytoplasm and dark precipitations in the cell wall after 3 weeks of Cr VI treatment. Electron energy loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) were applied to measure intracellular chromium distribution. Chromium was only detected after 3 weeks of 10μM Cr VI treatment in electron dense precipitations found in bag-like structures along the inner side of the cell walls together with iron and elevated levels of oxygen, pointing toward an accumulation respectively extrusion of chromium in form of an iron–oxygen compound. Atomic emission spectroscopy (EMS) revealed that Micrasterias cells are able to accumulate considerable amounts of chromium and iron. During chromium treatment the Cr:Fe ratio shifted in favor of chromium, which implied that chromium may be taken up instead of iron. Significant and rapid increase of ROS production within the first 5min of treatment confirms an active Cr VI uptake. SOD and CAT activity after Cr VI treatment did not show a response, while the glutathione pool determined by immuno-TEM decreased significantly in chromium treated cells, showing that glutathione is playing a major role in intracellular ROS and chromium detoxification.

RNA-Seq Analysis of Sulfur-Deprived Chlamydomonas Cells Reveals Aspects of Acclimation Critical for Cell Survival

The Chlamydomonas reinhardtii transcriptome was characterized from nutrient-replete and sulfur-depleted wild-type and snrk2.1 mutant cells. This mutant is null for the regulatory Ser-Thr kinase SNRK2.1, which is required for acclimation of the alga to sulfur deprivation. The transcriptome analyses used microarray hybridization and RNA-seq technology. Quantitative RT-PCR evaluation of the results obtained by these techniques showed that RNA-seq reports a larger dynamic range of expression levels than do microarray hybridizations. Transcripts responsive to sulfur deprivation included those encoding proteins involved in sulfur acquisition and assimilation, synthesis of sulfur-containing metabolites, Cys degradation, and sulfur recycling. Furthermore, we noted potential modifications of cellular structures during sulfur deprivation, including the cell wall and complexes associated with the photosynthetic apparatus. Moreover, the data suggest that sulfur-deprived cells accumulate proteins with fewer sulfur-containing amino acids. Most of the sulfur deprivation responses are controlled by the SNRK2.1 protein kinase. The snrk2.1 mutant exhibits a set of unique responses during both sulfur-replete and sulfur-depleted conditions that are not observed in wild-type cells; the inability of this mutant to acclimate to S deprivation probably leads to elevated levels of singlet oxygen and severe oxidative stress, which ultimately causes cell death. The transcriptome results for wild-type and mutant cells strongly suggest the occurrence of massive changes in cellular physiology and metabolism as cells become depleted for sulfur and reveal aspects of acclimation that are likely critical for cell survival.